2-alkylidene-aminoguanidines and methods of use therefor

ABSTRACT

The present invention relates to compositions and methods for inhibiting nonenzymatic cross-linking (protein aging) which contain 2-alkylidene aminoguanidines and derivatives thereof. Accordingly, a composition is disclosed which comprises an agent capable of inhibiting the formation of advanced glycosylation endproducts of target proteins by reacting with the carbonyl moiety of the early glycosylation product of such target proteins formed by their initial glycosylation. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated.

This invention was made in part with Government support under Grant No.PHS AM 19655 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present Application is a Continuation-In-Part of copendingapplication Ser. No. 07/605,654 filed Oct. 30, 1990, which is aContinuation-In-Part of U.S. Ser. No. 07/264,930, filed Nov. 2, 1988 andis now U.S. Pat. No. 4,983,604, which is a Continuation-In-Part ofU.S.S.N. 119,958, filed Nov. 13, 1987 which is now U.S. Patent No.4,908,446, which is a Continuation-In-Part of U.S.S.N. 798,032, filedNov. 14, 1985 and now U.S. Pat. No. 4,758,583, which is aContinuation-In-Part of U.S.S.N. 590,820, filed Mar. 19, 1984 and nowU.S. Pat. No. 4,665,192. Applicants claim the benefits of theseApplications under 35 U.S.C. §120.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to thesubject matter of the present invention: "COVALENT ATTACHMENT OF SOLUBLEPROTEINS BY NONENZYMATICALLY GLYCOSYLATED COLLAGEN: ROLE IN THE IN SITUFORMATION OF IMMUNE COMPLEXES", Brownlee et al., J. Exp. Med., 158, pp.1730-1744 (1983); and "AGING OF PROTEINS: ISOLATION AND IDENTIFICATIONOF FLUORESCENT CHROMOPHORE FROM THE REACTION OF POLYPEPTIDES WITHGLUCOSE", Pongor et al., Proc. Natl. Acad. Sci. USA, 81, pp. 2684-2688,(1984), and "ADVANCED GLYCOSYLATION ENDPRODUCTS IN TISSUE AND THEBIOCHEMICAL BASIS OF DIABETIC COMPLICATIONS", Brownlee et al., The NewEng. J. of Med., 318, pp. 1315-1321 (1988).

BACKGROUND OF THE INVENTION

The present invention relates generally to the aging of proteinsresulting from their reaction with glucose and other reducing sugars,and more particularly to the inhibition of the reaction ofnonenzymatically glycosylated proteins and the often resultant formationof advanced glycosylation endproducts and cross-links.

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food, which was identified by Maillard in 1912,who observed that glucose or other reducing sugars react with aminoacids to form adducts that undergo a series of dehydrations andrearrangements to form stable brown pigments. Maillard, C. R. Acad.Sci., 154. pp. 66-68, (1912). Further studies have suggested that storedand heat treated foods undergo nonenzymatic browning as a result of thereaction between glucose and the polypeptide chain, and that theproteins are resultingly cross-linked and correspondingly exhibitdecreased bioavailability.

This reaction between reducing sugars and food proteins was found tohave its parallel in vivo. Thus, the nonenzymatic reaction betweenglucose and the free amino groups on proteins to form a stable,1-deoxyketosyl adduct, known as the Amadori product, has been shown tooccur with hemoglobin, wherein a rearrangement of the amino terminal ofthe beta-chain of hemoglobin by reaction with glucose, forms the adductknown as hemoglobin A_(1c). The reaction has also been found to occurwith a variety of other body proteins, such as lens crystallins,collagen and nerve proteins. See Bunn et al., Biochem. Biophys. Res.Comm., 67,pp. 103-109 (1975); Koenig et al., J. Biol. Chem., 252. pp.2992-2997 (1977); Monnier et al., in Maillard Reaction in Food andNutrition, ed. Waller, G.A., American Chemical Society, 215, pp. 431-448(1983); and Monnier and Cerami, Clinics in Endocrinology and Metabolism,11, pp. 431-452 (1982).

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 to 90 years. See, Monnier et al., Science, 211. pp. 491-493(1981); Monnier et al., Biochem. Biophys. Acta, 760. pp. 97-103 (1983);and, Monnier et al., Proc. Natl. Acad. Sci., 81. pp. 583-587 (1984).Interestingly, the aging of collagen can be mimicked in vitro by thecross-linking induced by glucose; and the capture of other proteins andthe formation of adducts by collagen, also noted, is theorized to occurby a cross-linking reaction, and is believed to account for the observedaccumulation of albumin and antibodies in kidney basement membrane. See,Brownlee et al, J. Exp. Med., 158,pp. 1739-1744 (1983); and Kohn et al.,Diabetes. 33No. 1, pp. 57-59 (1984).

In Parent application Ser. No. 798,032, a method and associated agentswere disclosed that served to inhibit the formation of advancedglycosylation endproducts by reacting with the early glycosylationproduct that results from the original reaction between the targetprotein and glucose. Accordingly, inhibition was postulated to takeplace as the reaction between the inhibitor and the early glycosylationproduct appeared to interrupt the subsequent reaction of theglycosylated protein with additional protein material to form thecross-linked late stage product. One of the agents identified as aninhibitor was aminoguanidine, and the results of further testing haveborne out its efficacy in this regard.

While the success that has been achieved with aminoguanidine and similarcompounds is promising, a need continues to exist to identify anddevelop additional inhibitors that broaden the availability and perhapsthe scope of this potential activity and its diagnostic and therapeuticutility.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and compositions aredisclosed for the inhibition of the advanced glycosylation of proteins(protein aging). In particular, the compositions comprise agents forinhibiting nonenzymatic cross-linking (protein aging) due to theformation of advanced glycosylation endproducts. The agents may beselected from those materials capable of reacting with the earlyglycosylation product resulting from the reaction of glucose withproteins and preventing further reactions. Cross-linking caused by otherreactive sugars present in vivo or in foodstuffs, including ribose,galactose and fructose would also be prevented by the methods andcompositions of the present invention.

The agents comprise compounds having the following structural formula:##STR1## wherein R₁ is hydrogen or acyl;

R₂ is hydrogen or lower alkyl;

X is a substituent selected from the group consisting of lower alkyl,carboxy, carboxymethyl, or a phenyl or pyridyl group, optionallysubstituted by halogen, lower alkyl, hydroxy lower alkyl, hydroxy, oracetylamino with the proviso that when X is a phenyl or pyridyl group,optionally substituted, then R₂ is hydrogen; and their biocompatible andpharmaceutically acceptable acid addition salts, and mixtures thereof;and a carrier therefor.

The compounds utilized in the compositions of this invention appear toreact with the early glycosylation product thereby preventing the samefrom later forming the advanced glycosylation end products which lead toprotein cross-links, and thereby, to protein aging.

The present invention also relates to a method for inhibiting proteinaging by contacting the initially glycosylated protein at the stage ofthe early glycosylation product with a quantity of one or more of theagents of the present invention, or a composition containing the same.In the instance where the present method has industrial application, oneor more of the agents may be applied to the proteins in question, eitherby introduction into a mixture of the same in the instance of a proteinextract, or by application or introduction into foodstuffs containingthe protein or proteins, all to prevent premature aging and spoilage ofthe particular foodstuffs.

The ability to inhibit the formation of advanced glycosylationendproducts carries with it significant implications in all applicationswhere protein aging is a serious detriment. Thus, in the area of foodtechnology, the retardation of food spoilage would confer an obviouseconomic and social benefit by making certain foods of marginalstability less perishable and therefore more available for consumers.Spoilage would be reduced as would the expense of inspection, removal,and replacement, and the extended availability of the foods could aid instabilizing their price in the marketplace.

Similarly, in other industrial applications where the perishability ofproteins is a problem, the admixture of the agents of the presentinvention in compositions containing such proteins would facilitate theextended useful life of the same. Presently used food preservatives anddiscoloration preventatives such as sulfur dioxide, known to causetoxicity including allergy and asthma in animals, can be replaced withcompounds such as those described herein.

The present method has particular therapeutic application as theMaillard process acutely affects several of the significant proteinmasses in the body, among them collagen, elastin, lens proteins, and thekidney glomerular basement membranes. These proteins deteriorate bothwith age (hence the application of the term "protein aging") and aconsequence of diabetes. Accordingly, the ability to either retard orsubstantially inhibit the formation of advanced glycosylationendproducts carries the promise of treatment for diabetes and of course,improving the quality and, perhaps, duration of animal life.

The present agents are also useful in the area of personal appearanceand hygiene, as they prevent the staining of teeth by cationicanti-microbial agents with anti-plaque properties, such aschlorhexidine.

Accordingly, it is a principal object of the present invention toprovide a method for inhibiting the extensive cross-linking of proteinsthat occurs as an ultimate consequence of the reaction of the proteinswith glucose and other reactive sugars, by correspondingly inhibitingthe formation of advanced glycosylation endproducts.

It is a further object of the present invention to provide a method asaforesaid which is characterized by a reaction with an initiallyglycosylated protein identified as an early glycosylation product.

It is a further object of the present invention to provide a method asaforesaid which prevents the rearrangement and cross-linking of the saidearly glycosylation products to form the said advanced glycosylationendproducts.

It is a yet further object of the present invention to provide agentscapable of participating in the reaction with the said earlyglycosylation products in the method as aforesaid.

It is a still further object of the present invention to providetherapeutic methods of treating the adverse consequences of proteinaging by resort to the aforesaid method and agents.

It is a still further object of the present invention to provide amethod of inhibiting the discoloration of teeth by resort to theaforesaid method and agents.

It is a still further object of the present invention to providecompositions including pharmaceutical compositions, all incorporatingthe agents of the present invention.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, agents, compositions includingpharmaceutical compositions containing said agents and associatedmethods have been developed which are believed to inhibit the formationof advanced glycosylation endproducts in a number of target proteinsexisting in both animals and plant material. In particular, theinvention relates to a composition which may contain one or more agentscomprising compounds having the structural formula ##STR2## wherein R₁is hydrogen or acyl;

R₂ is hydrogen or lower alkyl;

X is a substituent selected from the group consisting of lower alkyl,carboxy, carboxymethyl, or a phenyl or pyridyl group, optionallysubstituted by halogen, lower alkyl, hydroxy lower alkyl, hydroxy, oracetylamino with the proviso that when X is a phenyl or pyridyl group,optionally substituted, then R₂ is hydrogen; and their biocompatible andpharmaceutically acceptable acid addition salts, and mixtures thereof;and a carrier therefor.

The lower alkyl groups referred to herein contain 1-6 carbon atoms andinclude methyl, ethyl, propyl, butyl, pentyl, hexyl, and thecorresponding branched chain isomers thereof. The halo variants can befluoro, chloro, bromo or iodo substituents.

Equivalent to the compounds of Formula I for the purpose of thisinvention are the biocompatible and pharmaceutically acceptable saltsthereof. Such salts can be derived from a variety of organic andinorganic acids including but not limited to methanesulfonic,hydrochloric, toluenesulfonic, sulfuric, maleic, acetic and phosphoricacids.

Certain of the compounds of Formula I are novel compounds. The novelcompounds are those wherein X is a carboxymethyl group and R₁ and R₂ areas hereinbefore defined.

Of the compounds encompassed by Formula I, certain substituents arepreferred. For instance, R₁ is preferably a methyl group and X ispreferably a phenyl or substituted phenyl group.

Representative compounds of the present invention are:

N-acetyl-2-(phenylmethylene)hydrazinecarboximidamide;

2-(phenylmethylene)hydrazinecarboximidamide;

2-(2,6-dichlorophenylmethylene)hydrazinecarboximidamide pyridoxalguanylhydrazone;

pyridoxal phosphate guanylhydrazone;

2-(1-methylethylidene)hydrazinecarboximidamide;

pyruvic acid guanylhydrazone;

4-acetamidobenzaldehyde guanylhydrazone;

4-acetamidobenzaldehyde N-acetylguanylhydrazone;

acetoacetic acid guanylhydrazone;

and the biocompatible and pharmaceutically acceptable salts thereof.

The above compounds are capable of inhibiting the formation of advancedglycosylation endproducts on target proteins. The cross-linking of theprotein to form the advanced glycosylation endproduct contributes to theentrapment of other proteins and results in the development in vivo ofconditions such as reduced elasticity and wrinkling of the skin, certainkidney diseases, atherosclerosis, osteoarthritis and the like.Similarly, plant material that undergoes nonenzymatic browningdeteriorates and, in the case of foodstuffs, become spoiled or toughenedand, consequently, inedible. Thus, the compounds employed in accordancewith this invention inhibit this late stage Maillard effect andintervene in the deleterious changes described above.

The rationale of the present invention is to use agents which block thepost-glycosylation step, i.e., the formation of fluorescent chromophoressuch as that identified in Pongor, et al., supra and Farmar et al.,supra, among others, the presence of which chromophores is associatedwith, and leads to adverse sequelae of diabetes and aging. An idealagent would prevent the formation of the chromophore and its associatecross-links of proteins to proteins and trapping of proteins on theother proteins, such as occurs in arteries and in the kidney.

The chemical nature of the early glycosylation products with which thecompounds of the present invention are believed to react, isspeculative. Early glycosylation products with carbonyl moieties thatare involved in the formation of advanced glycosylation endproducts, andthat may be blocked by reaction with the compounds of the presentinvention, have been postulated. In one case, the reactive carbonylmoieties of Amadori products or their further condensation, dehydrationand/or rearrangement products, may condense to form advancedglycosylation endproducts. Another proposed mechanism is the formationof reactive carbonyl compounds, containing one or more carbonyl moieties(such as glycolaldehyde, glyceraldehyde or 3-deoxyglucosone) from thecleavage of Amadori or other early glycosylation endproducts (see, forexample, Gottschalk, A. (1972) in The Glycoproteins (Gottschalk, A., ed)Part A, pp. 141-157, Elsevier Publishing Co., New York; Reynolds, T.M.(1965) Adv. Food Res., 14, pp. 167-283), and by subsequent reactionswith an amine or Amadori product to form carbonyl containing advancedglycosylation products such as the alkylformylglycosylpyrroles describedby Farmar et al, supra.

Several investigators have studied the mechanism of advancedglycosylation product formation. In vitro studies by Eble et al.,(1983), "Nonenzymatic Glucosylation and Glucose-dependent Cross-linkingof Protein", J. Biol. Chem., 258:9406-9412, concerned the cross-linkingof glycosylated protein with nonglycosylated protein in the absence ofglucose. Eble et al. sought to elucidate the mechanism of the Maillardreaction and accordingly conducted controlled initial glycosylation ofRNAase as a model system, which was then examined under varyingconditions. In one aspect, the glycosylated protein material wasisolated and placed in a glucose-free environment and thereby observedto determine the extent of cross-linking.

Eble et al. thereby observed that cross-linking continued to occur notonly with the glycosylated protein but with non-glycosylated proteins aswell. One of the observations noted by Eble et al. was that the reactionbetween glycosylated protein and the protein material appeared to occurat the location on the protein chain of the amino acid lysine.Confirmatory experimentation conducted by Eble et al. in this connectiondemonstrated that free lysine would compete with the lysine on RNAasefor the binding of glycosylated protein. Thus, it might be inferred fromthese data that lysine may serve as an inhibitor of advancedglycosylation; however, this conclusion and the underlying observationsleading to it should be taken in the relatively limited context of themodel system prepared and examined by Eble et al. Clearly, Eble et al.does not appreciate, nor is there a suggestion therein, of thediscoveries that underlie the present invention, with respect to theinhibition of advanced glycosylation of proteins both in vitro and invivo.

The experiments of Eble et al. do not suggest the reactive cleavageproduct mechanism or any other mechanism in the in vivo formation ofadvanced glycosylation endproducts in which glucose is always present.In fact, other investigators support this mechanism to explain theformation of advanced glycosylated endproducts in vivo (see for exampleHayase et al, 1989, supra; Sell and Monnier, 1989, supra; Oimomi et al.,Agric. Biol. Chem., 53(6):1727-1728 (1989); and Diabetes Research andClinical Practice, 6:311-313 (1989). Accordingly, the use of lysine asan inhibitor in the Eble et al. model system has no bearing upon theutility of the compounds of the present invention in the inhibition ofadvanced glycosylated endproducts formation in the presence of glucosein vivo. and the amelioration of complications of diabetes and aging.

The compositions useful in the present invention comprise or containagents capable of reacting with the active carbonyl intermediate of anearly glycosylation product. Suitable agents are the compounds ofFormula I of the present invention.

The present invention likewise relates to methods for inhibiting theformation of advanced glycosylation endproducts, which comprisecontacting the target proteins with a composition of the presentinvention. In the instance where the target proteins are contained infoodstuffs, whether of plant or animal origin, these foodstuffs couldhave applied to them by various conventional means a compositioncontaining the present agents.

In the food industry, sulfites were found years ago to inhibit theMaillard reaction and are commonly used in processed and stored foods.Recently, however, sulfites in food have been implicated in severe andeven fatal reactions in asthmatics. As a consequence, the sulfitetreatment of fresh fruits and vegetables has been banned. The mechanismfor the allergic reaction is not known. Accordingly, the presentcompositions and agents offer a nontoxic alternative to sulfites in thetreatment of foods in this manner.

As is apparent from a discussion of the environment of the presentinvention, the present methods and compositions hold the promise forarresting the aging of key proteins both in animals and plants, andconcomitantly, conferring both economic and medical benefits as a resultthereof. In the instance of foodstuffs, the administration of thepresent composition holds the promise for retarding food spoilagethereby making foodstuffs of increased shelf life and greateravailability to consumers. Replacement of currently-used preservatives,such as sulfur dioxide known to cause allergies and asthma in humans,with non-toxic, biocompatible compounds is a further advantage of thepresent invention.

The therapeutic implications of the present invention relate to thearrest of the aging process which has, as indicated earlier, beenidentified in the aging of key proteins by advanced glycosylation andcross-linking. Thus, body proteins, and particularly structural bodyproteins, such as collagen, elastin, lens proteins, nerve proteins,kidney glomerular basement membranes and other extravascular matrixcomponents would all benefit in their longevity and operation from thepractice of the present invention. The present invention thus reducesthe incidence of pathologies involving the entrapment of proteins bycross-linked target proteins, such as retinopathy, cataracts, diabetickidney disease, glomerulosclerosis, peripheral vascular disease,arteriosclerosis obliterans, peripheral neuropathy, stroke,hypertension, atherosclerosis, osteoarthritis, periarticular rigidity,loss of elasticity and wrinkling of skin, stiffening of joints,glomerulonephritis, etc. Likewise, all of these conditions are inevidence in patients afflicted with diabetes mellitus. Thus, the presenttherapeutic method is relevant to treatment of the noted conditions inpatients either of advanced age or those suffering from one of thementioned pathologies.

Protein cross-linking through advanced glycosylation product formationcan decrease solubility of structural proteins such as collagen invessel walls (see Brownlee et al., Science. 232, pp. 1629-1632, (1986)),and can also trap serum proteins, such as lipoproteins to the collagen.Also, this may result in increased permeability of the endothelium andconsequently covalent trapping of extravasated plasma proteins insubendothelial matrix, and reduction in susceptibility of both plasmaand matrix proteins to physiologic degradation by enzymes. (See Brownleeet al., Diabetes, 35, Suppl. 1, p. 42A (1986)). For these reasons, theprogressive occlusion of diabetic vessels induced by chronichyperglycemia has been hypothesized to result from excessive formationof glucose-derived cross-links. Such diabetic macrovascular changes andmicrovascular occlusion can be effectively prevented by chemicalinhibition of advanced glycosylation product formation utilizing acomposition and the methods of the present invention.

Studies indicate that the development of chronic diabetic damage intarget organs is primarily linked to hyperglycemia so that tightmetabolic control would delay or even prevent end-organ damage. SeeNicholls et al., Lab. Invest., 60, No. 4, p. 486 (1989), which discussesthe effects of islet isografting and aminoguanidine in murine diabeticnephropathy. These studies further evidence that aminoguanidinediminishes aortic wall protein cross-linking in diabetic rats andconfirm earlier studies by Brownlee et al., Science, 232, pp. 1629-1632(1986) to this additional target organ of complication of diabetes.Also, an additional study showed the reduction of immunoglobulintrapping in the kidney by aminoguanidine (Brownlee et al., Diabetes, 35,Suppl. 1, p. 42A (1986)).

Further evidence in the streptozotocin-diabetic rat model thataminoguanidine administration intervenes in the development of diabeticnephropathy was presented by Brownlee et al., 1988, supra, with regardto morphologic changes in the kidney which are hallmarks of diabeticrenal disease. These investigators reported that the increasedglomerular basement membrane thickness, a major structural abnormalitycharacteristic of diabetic renal disease, was prevented withaminoguanidine.

Taken together, these data strongly suggest that inhibition of theformation of advanced glycosylation endproducts (AGEs), by the teachingof the present invention, may prevent late, as well as early, structurallesions due to diabetes, as well as changes during aging caused by theformation of AGE's.

Diabetes-induced changes in the deformability of red blood cells,leading to more rigid cell membranes, is another manifestation ofcross-linking and aminoguanidine has been shown to prevent it in vivo.In such studies, New Zealand White rabbits, with induced, long-termdiabetes are used to study the effects of a test compound on red bloodcell (RBC) deformability (df). The test compound is administered at arate of 100 mg/kg by oral gavage to diabetic rabbits (Brown et al.,Presentation of Abstract for Association for Academic MinorityPhysicians, Annual Scientific Meeting (1989)).

Increased cross-linking of collagen in diabetic rats has shown to beprevented by aminoguanidine. Oxlund and Andreassen, "The increase inbiochemical and biomechanical stability of collagen in diabetic rats isprevented by aminoguanidine treatment", European Association for theStudy of Diabetes, Twenty-fifth Annual Meeting, p. 525A, Abstract No.371, 1989 showed the effect when thermal stability of tendon fibers wasassessed by breaking time in a urea bath, as well as mechanicalstrength. Soulis et al., "Aminoguanidine reduces tissue fluorescence butnot albuminuria in diabetic rats". NIH Conference on the MaillardReaction in Aging, Diabetes, and Nutrition, Bethesda, Md., Sep. 22-23,1988, page 30) showed the same effect on collagen in the aorta, measuredby fluorescence and solubility.

Giambione and Brownlee, "Aminoguanidine Treatment Normalizes IncreasedSteady-state Levels of Laminin B1 mRNA in Kidneys of Long-termStreptozotocin-diabetic Rats" Diabetes, 38,Supplement 2:83A Forty-ninthAnnual Meeting, American Diabetes Association (1989) showed thataminoguanidine treatment to diabetic rats prevents the diabetes-inducedincrease in laminin B₁ mRNA in the kidney. This indicates thataminoguanidine may prevent overproduction of matrix, which leads tobasement membrane thickening and morphologic and functionaldeterioration of vasculature in kidneys and other organs.

A further consequence of diabetes is the hyperglycemia-induced matrixbone differentiation resulting in decreased bone formation usuallyassociated with chronic diabetes. In animal models, diabetes reducesmatrix-induced bone differentiation by 70% (Am. J. Phys., 238 (1980)).

In the instance where the compositions of the present invention areutilized for in vivo or therapeutic purposes, it may be noted that thecompounds or agents used therein are biocompatible. Pharmaceuticalcompositions may be prepared with a therapeutically effective quantityof the agents or compounds of the present invention and may include apharmaceutically acceptable carrier, selected from known materialsutilized for this purpose. Such compositions may be prepared in avariety of forms, depending on the method of administration. Also,various pharmaceutically acceptable addition salts of the compounds ofFormula I may be utilized.

A liquid form would be utilized in the instance where administration isby intravenous, intramuscular or intraperitoneal injection. Whenappropriate, solid dosage forms such as tablets, capsules, or liquiddosage formulations such as solutions and suspensions, etc., may beprepared for oral administration. For topical or dermal application tothe skin or eye, a solution, a lotion or ointment may be formulated withthe agent in a suitable vehicle such as water, ethanol, propyleneglycol, perhaps including a carrier to aid in penetration into the skinor eye. For example, a topical preparation could include up to about 10%of the compound of Formula I. Other suitable forms for administration toother body tissues are also contemplated.

In the instance where the present method has therapeutic application,the animal host intended for treatment may have administered to it aquantity of one or more of the agents, in a suitable pharmaceuticalform. Administration may be accomplished by known techniques, such asoral, topical and parenteral techniques such as intradermal,subcutaneous, intravenous or intraperitoneal injection, as well as byother conventional means. Administration of the agents may take placeover an extended period of time at a dosage level of, for example, up toabout 25 mg/kg.

As noted earlier, the invention also extends to a method of inhibitingthe discoloration of teeth resulting from nonenzymatic browning in theoral cavity which comprises administration to a subject in need of suchtherapy an amount effective to inhibit the formation of advancedglycosylation endproducts of a composition comprising an agent of thestructural Formula I.

The nonenzymatic browning reaction which occurs in the oral cavityresults in the discoloration of teeth. Presently used anti-plaque agentsaccelerate this nonenzymatic browning reaction and further the stainingof the teeth. Recently, a class of cationic anti-microbial agents withremarkable anti-plaque properties have been formulated in oral rinsesfor regular use to kill bacteria in the mouth. These agents, thecationic antiseptics, include such agents as alexidine, cetyl pyridiniumchloride, chlorhexidine gluconate, hexetidine, and benzalkoniumchloride.

Tooth staining by chlorhexidine and other anti-plaque agents apparentlyresults from the enhancement of the Maillard reaction. Nordbo, J. Dent.Res., 58, p. 1429 (1979) reported that chlorhexidine and benzalkoniumchloride catalyze browning reactions in vitro. Chlorhexidine added tomixtures containing a sugar derivative and a source of amino groupsunderwent increased color formation, attributed to the Maillardreaction. It is also known that use of chlorhexidine results in anincreased dental pellicle. Nordbo proposed that chlorhexidine resultedin tooth staining in two ways: first, by increasing formation ofpellicle which contains more amino groups, and secondly, by catalysis ofthe Maillard reaction leading to colored products.

In accordance with this method, the compounds of Formula I areformulated into compositions adapted for use in the oral cavity.Particularly suitable formulations are oral rinses and toothpastesincorporating the active agent.

In the practice of this invention, conventional formulating techniquesare utilized with nontoxic, pharmaceutically acceptable carrierstypically utilized in the amounts and combinations that are well-knownfor the formulation of such oral rinses and toothpastes.

The agent of Formula I is formulated in compositions in an amounteffective to inhibit the formation of advanced glycosylationendproducts. This amount will, of course, vary with the particular agentbeing utilized and the particular dosage form, but typically is in therange of 0.01% to 1.0%, by weight, of the particular formulation.

Additionally, since the agents of the aforesaid method are concentratedin the salivary glands upon oral ingestion or parenteral administration,they can be so administered. This concentration in the salivary glandsresults in their secretion into saliva, the net result being that theyare functionally placed in the oral cavity where they can effect theirdesired method. For such administration, the particular agent can beformulated in any conventional oral or parenteral dosage form. Aparticularly desirable dosage form is the incorporation of the agentinto a vitamin tablet or fluoride tablet so as to maximize patient, andparticularly juvenile patient, compliance.

Certain of the compounds of Formula I of the present invention are knownin the art. For instance,N-acetyl2-phenylmethylene)hydrazinecarboximidamide is described in Latv.PSR Zinat. Akad. Vestis, Kim Ser. (3), 347-351(1969); benzaldehydeguanylhydrazone acetate in C.A. 100:85533 g; 2-(2,6-dichlorophenylmethylene)hydrazine carboximide amide (guanabenz acetate) in BritishPat. No. 1,019,120 and German Pat. No. 1,804,634;4-acetamidobenzaldehyde guanylhydrazone hydrochloride in Chem. Pharm.Bull., 22(10):2444-7 (1974); pyridoxyl guanylhydrazone dihydrochloridein J. Med. Pharm. Chem., 5, 1367, 1370 (1962); pyridoxal phosphateguanylhydrazone hydrochloride in Arch. Ital. Prtol. Clin. Tumori,8:33-44 (1965); 2-(1-methyethylidene)hydrazinecarboximidamidemonohydrochloride in J. Am. Chem. Soc., 74:2981 (1952); pyruvic acidguanylhydrazone hydrochloride in Chem. Pharm. Bull., 12:100103 (1964).Salicaldehyde hydrazone is available from Aldrich Chemical Company.These and other variants of Formula I are available from variouschemical supply houses or readily synthesizable from the chemicalliterature. Certain of the compounds of Formula I are novel compounds,and they are described hereinbelow.

For instance, the compound of Formula I wherein R₁ is hydrogen, R₂ is amethyl group and X is carboxymethylene group is a novel compound whichcan exist in tautomeric form, i.e., ##STR3## This compound is typicallyprepared by reaction of lithium acetoacetate with aminoguanidine atreflux temperatures.

The compounds of Formula I wherein R₁ is an acyl group can beconveniently prepared from the corresponding compound wherein R₁ ishydrogen. The compound of Formula I wherein R₁ is hydrogen is acylatedby conventional techniques, typically by contact with the appropriateacyl halide or acyl anhydride in a polar solvent in the presence of anacid acceptor. Pyridine is often the solvent of choice and triethyleneamine the acid acceptor, but these can be varied depending upon thereaction conditions necessary.

The compounds of Formula I wherein X is an acetylaminophenyl substituentare typically prepared by reaction of the appropriateacetamidobenzaldehyde (o, m or p substituent pattern) withaminoguanidine to form the acetamidobenzaldehyde guanyl hydrazone (theFormula I compound wherein R₁ =hydrogen). This compound can then befurther reacted as described above to form the corresponding compoundwherein R₁ is an acyl group.

The following examples are illustrative of the compositions and methodsof the present invention.

EXAMPLE 1 ACETOACETIC ACID QUANYLHYDRAZONE INNER SALT

Lithium acetoacetate (0.655 g) in 2 ml water was treated with 0.665 gaminoguanidine HCl in 1.33 ml water. The solution was heated to refluxover 30 minutes and then allowed to cool. Ethanol (10 ml) was added andthe mixture was stored at -20° C. for 24 hrs., then at 4° C. for 24 hrs.The mixture was allowed to reach room temperature and filtered. Thefiltrate was concentrated to a paste and triturated with 5 ml ethanol.Filtration and washing with ethanol gave 0.522 to the hydrazone as amicrocrystalline powder, melting point 138.6°-140° C.

EXAMPLE 2

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit glucose-mediated development offluorescence of bovine serum albumin (BSA), a measure of cross-linking.Compounds were incubated under aseptic conditions at a concentration of1 mM with 400 mM glucose and 100 mg/mL BSA in a 1.5 M sodium phosphatebuffer, pH 7.4.

Samples of the incubation mixture were taken immediately and after 1week incubation at 37° C. for measurement of fluorescence. For each testcompound, control incubations in buffer were made of compound alone (C),compound plus glucose (G+C), and compound plus BSA (B+C). An additionalset of incubations of glucose and BSA (B+G) were prepared as thebaseline controls against which were measured the ability of thecompounds to inhibit. Each incubation was made in triplicate.

Fluorescence (excitation, 370 nm; emission, 440 nm) was measured on eachsample after a 100-fold dilution in distilled water.

The % inhibition of browning of each test compound was calculated asfollows. Each F represents the fluorescence measurement of that sampleafter 1 week incubation less its fluorescence before incubation.##EQU1## where B=BSA, G=glucose, and C-test compound.

Percent inhibition of browning by various test compounds at 1 mM:

0%: no inhibitor

38.7: N-acetyl-2-(phenylmethylene)hydrazinecarboximidamide;

19.7 : 2-(2,6-dichlorophenylmethylene)hydrazinecarboximidamidemonoacetate;

12.7: pyridoxal phosphate guanylhydrazone dihydrochloride;

30.5: 2-(1-methylethylidene)hydrazinecarboximidamide monohydrochloride;

50.8: pyruvic acid guanylhydrazone hydrochloride:

54.2: salicaldehyde hydrazone.

The above experiments suggest that this type of drug therapy may havebenefit in reducing the pathology associated with the advancedglycosylation of proteins and the formation of cross-links betweenproteins and other macromolecules. Drug therapy may be used to preventthe increased trapping and cross-linking of proteins that occurs indiabetes and aging which leads to sequelae such as retinal damage, andextra-vascularly, damage to tendons, ligaments and other joints. Thistherapy might retard atherosclerosis and connective tissue changes thatoccur with diabetes and aging. Both topical, oral, and parenteral routesof administration to provide therapy locally and systemically arecontemplated.

EXAMPLE 3

    ______________________________________                                        Tablet            mg/tablet                                                   ______________________________________                                        Compound of Formula I                                                                           50                                                          Starch            50                                                          Mannitol          75                                                          Magnesium stearate                                                                               2                                                          Stearic acid       5                                                          ______________________________________                                    

The compound, a portion of the starch and the lactose are combined andwet granulated with starch paste. The wet granulation is placed on traysand allowed to dry overnight at a temperature of 45° C. The driedgranulation is comminuted in a comminutor to a particle size ofapproximately 20 mesh. Magnesium stearate, stearic acid and the balanceof the starch are added and the entire mix blended prior to compressionon a suitable tablet press. The tablets are compressed at a weight of232 mg. using a 11/32' punch with a hardness of 4 kg. These tablets willdisintegrate within a half hour according to the method described in USPXVI.

EXAMPLE 4

    ______________________________________                                        Lotion            mg/g                                                        ______________________________________                                        Compound of Formula I                                                                            1.0                                                        Ethyl alcohol     400.0                                                       Polyethylene glycol 400                                                                         300.0                                                       Hydroxypropyl cellulose                                                                          5.0                                                        Propylene glycol  to make 1.0 g                                               ______________________________________                                    

EXAMPLE 5

    ______________________________________                                        Oral Rinse                                                                    ______________________________________                                        Compound of Formula I:  1.4%                                                  Chlorhexidine gluconate 0.12%                                                 Ethanol                 11.6%                                                 Sodium saccharin        0.15%                                                 FD&C Blue No. 1         0.001%                                                Peppermint Oil          0.5%                                                  Glycerine               10.0%                                                 Tween 60                0.3%                                                  Water to                100%                                                  ______________________________________                                    

EXAMPLE 6

    ______________________________________                                        Toothpaste                                                                    ______________________________________                                        Compound of Formula I:    5.5%                                                Sorbitol, 70% in water    25%                                                 Sodium saccharin          0.15%                                               Sodium lauryl sulfate     1.75%                                               Carbopol 934, 6% dispersion in water                                                                    15%                                                 Oil of Spearmint          1.0%                                                Sodium hydroxide, 50% in water                                                                          0.76%                                               Dibasic calcium phosphate dihydrate                                                                     45%                                                 Water to                  100%                                                ______________________________________                                    

EXAMPLE 7

To further study the ability of inhibitors of nonenzymatic browning toprevent the discoloration of protein on a surface, such as that whichoccurs on the tooth surface, the following surface browning experimentis performed. As a substitute for a pellicle-covered tooth surface,unexposed and developed photographic paper is used to provide a fixedprotein (gelatin, i.e., collagen) surface on a paper backing. Fivemillimeter circles are punched and immersed for one week at 50° C. in asolution of 100 mM glucose-6-phosphate in a 0.5 M phosphate buffer, pH7.4, containing 3 mM sodium azide. Glucose-6-phosphate is a sugarcapable of participating in nonenzymatic browning at a more rapid ratethan glucose. In addition to the glucose-6-phosphate, chlorhexidineand/or a compound of Formula I are included. After incubation, thegelatin/paper disks are rinsed with water, observed for brown color, andphotographed.

Incubation of the disks in glucose-6-phosphate alone shows slight browncolor versus disks soaked in buffer alone. Inclusion of chlorhexidine(in the form of Peridex® at a final concentration of 0.04%chlorhexidine) shows significant browning. Addition of a compound ofFormula I to the chlorhexidine inhibits browning of the gelatin, as doesinclusion of a compound of Formula I in the absence of chlorhexidine.

The slight brown color formed by the action of glucose-6-phosphate onthe gelatin surface alone and its prevention by a compound of Formula Idemonstrates the utility of the present invention in preventingnonenzymatic browning of tooth surfaces. The enhanced browning in thepresence of chlorhexidine and its prevention with a compound of FormulaI demonstrates the utility of the present invention in preventing theanti-plaque agent-enhanced nonenzymatic browning which occurs withchlorhexidine.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A method for inhibiting the advancedglycosylation of a target protein comprising contacting the targetprotein with an effective amount of composition comprising a compoundselected from the group consisting of compounds of the formula ##STR4##wherein R₁ is hydrogen or acyl;R₂ is hydrogen or lower alkyl; X is asubstituent selected from the group consisting of lower alkyl, carboxy,carboxymethyl, or a phenyl or pyridyl group, optionally substituted byhalogen, lower alkyl, hydroxy lower alkyl, hydroxy, or acetylamino withthe proviso that when X is a phenyl or pyridyl group, optionallysubstituted, then R₂ is hydrogen; and their biocompatible andpharmaceutically acceptable acid addition salts, and mixtures thereof;and a pharmaceutically acceptable carrier therefor.
 2. The method ofclaim 1 wherein said compound has the formula wherein X is hydrogen orphenyl or substituted phenyl group.
 3. The method of claim 1 whereinsaid compound is N-acetyl-2-(phenylmethylene)hydrazinecarboximidamide ora pharmaceutically acceptable salt thereof.
 4. The method of claim 1wherein said compound is 2-(phenylmethylene)hydrazinecarboximidamidemonoacetate or a pharmaceutically acceptable salt thereof.
 5. The methodof claim 1 wherein said compound is2-(2,6-dichlorophenylmethylene)hydrazinecarboximidamide monoacetate or apharmaceutically acceptable salt thereof.
 6. The method of claim 1wherein said compound is 4-acetamidobenzaldehyde guanylhydrazonehydrochloride or a pharmaceutically acceptable salt thereof.
 7. Themethod of claim 1 wherein said compound is 4-acetamidobenzaldehydeN-acetylguanylhydrazone or another pharmaceutically acceptable saltthereof.
 8. The method of claim 1 wherein said compound has the formulawherein X is a pyridyl or substituted pyridyl group.
 9. The method ofclaim 1 wherein said compound is pyridoxal guanylhydrazonedihydrochloride or a pharmaceutically acceptable salt thereof.
 10. Themethod of claim 1 wherein said compound is pyridoxal phosphateguanylhydrazone hydrochloride or a pharmaceutically acceptable saltthereof.
 11. The method of claim 1 wherein said compound has the formulawherein X is a carboxy group.
 12. The method of claim 1 wherein saidcompound is pyruvic acid guanylhydrazone hydrochloride or apharmaceutically acceptable salt thereof.
 13. The method of claim 1wherein said compound has the formula wherein X is a lower alkyl group.14. The method of claim 1 wherein said compound is2-(1-ethylethylidene)hydrazinecarboximidamide monohydrochloride or apharmaceutically acceptable salt thereof.
 15. The method of claim 1wherein said compound has the formula wherein X is a carboxymethylgroup.
 16. The method of claim 1 wherein said compound is acetoaceticacid guanylhydrazone or a pharmaceutically acceptable salt thereof.